The goal of this work is to understand the molecular mechanisms that regulate a fundamental aspect of the cell cycle - the dependency of the duplication of a cell's genome on the completion of the previous mitosis. We will focus our study on the only eukaryotic organsim Saccharomyces cerevisiae which has a well defined origin. It is therefore amenable to studies which seek to determine factors which modulate origin usage during the cell cycle. Our research plan builds on three recent links we have developed. The first between the mitotic kinase Cdc5p which is required to complete mitosis and the G1/S factor Dbf4p. The second between Dbf4p and the MCM proteins which are required to license replication to once per cell cycle. The third between the APC and the degredation of Cdc5p and Dbf4p late in Mitosis. We have four specific aims. SA#1: Determine the cell cycle regulated interactions of Dbf4p with chromatin and origins. SA#2: Determine the factors that regulate the function of Dbf4p during the cell cycle. SA#3: Identify the role for the kinase Cdc5p in mitosis and replication. SA#4: Identify and functionally characterize novel proteins that interact with or regulate Dbf4p. Through this research we hope to clarify aspects of the ways in which cells separate the activities responsible for priming origins during M/G1, from those which activate initiation of DNA replication during G1/S. Such partitioning of cellular labour may help prevent re- replication prior to the completion of mitotis by requiring that the factors which direct the final steps of mitosis are also responsible for the completion of the first steps leading to the initiation of DNA replication.